The formation of pituitary hormones and active peptides from higher molecular weight precursors is dependent upon the action of specific endopeptidases. Degradation of hormones and peptide-factors controlling pituitary function is also dependent on the activity of such enzymes. The nature of these enzymes remains to be established. We have recently succeeded, using model synthetic substrates, in identifying and purifying three apparently new pituitary endopeptidases. We have also obtained data suggesting the presence of other endopeptidases in pituitary extracts. The three enzymes include a "cathepsin B-like" activity highly specific toward the sequence X-Leu-Arg appears as a precipitate X' or X-Leu-Leu-Arg appears as a precipitate X' (X' equals chromogenic group), a Na ion-inhibited neutral endopeptidase and a prolyl endopeptidase. We propose to purify these enzymes to homogeneity and to study their biochemical properties. The other endopeptidases will also be purified and characterized. Special effort will be directed toward determination of the specificity of the enzymes using natural substrates. The beta-lipotropin molecule will be used as a model to determine the possible function of these enzymes in the interconversions of this prohormone. Specificity toward other biologically active peptides such as beta-endorphin, luteinizing hormone-releasing hormone, somatostatin, oxytocin and vasopressin will be examined. Substrates synthesized in our laboratory will be employed for the histochemical localization of the enzymes. Immunohistochemical localization with specific antibodies obtained against the isolated endopeptidases is also planned. Our methods allow determination of endopeptidase activities in minute samples of pituitary tissue. These procedures will be used to detect any changes in enzyme activity related to changes in the secretory status of the pituitary induced by accepted procedures. The study should provide information relevant to the evaluation of the physiological role of pituitary endopeptidases.